Progress of Marine Biofouling and Antifouling Technologies

Review

Materials Science March 2011 Vol.56 No.7: 598612 doi: 10.1007/s11434-010-4158-4

SPECIAL TOPICS:

Progress of marine biofouling and antifouling technologies

CAO Shan, WANG JiaDao*, CHEN HaoSheng & CHEN DaRong

State Key Laboratory of Tribology, Tsinghua University, Beijing 100084, China

Received April 13, 2010; accepted July 22, 2010; published online November 26, 2010

Adhesion of marine fouling organisms on artificial surfaces such as ship hulls causes many problems, including extra energy consumption, high maintenance costs, and increased corrosion. Therefore, marine antifouling is an important issue. In this review, physical and biochemical developments in the field of marine biofouling, which involves biofilm formation and macro-organism settlement, are discussed. The major antifouling technologies based on traditional chemical methods, biological methods, and physical methods are presented. The chemical methods include self-polishing types such as tributyltin (TBT) self-polishing copolymer coatings, which despite its good performance has been banned since 2008 because of its serious environmental impact. Therefore, other methods have been encouraged. These include coatings with copper compounds and biocide boosters to replace the TBT coatings. Biological extracts of secreted metabolites and enzymes are anticipated to act as antifoulants. Physical methods such as modification of surface topography, hydrophobic properties, and charge potential have also been considered to prevent biofouling. In this review, most of the current antifouling technologies are discussed. It is proposed that the physical antifouling technologies will be the ultimate antifouling solution, because of their broad-spectrum effectiveness and zero toxicity. biofouling, antifouling technology, biofilm, adhesion mechanism

Citation: Cao S, Wang J D, Chen H S, et al. Progress of marine biofouling and antifouling technologies. Chinese Sci Bull, 2011, 56: 598–612, doi: 10.1007/s11434-010-4158-4

In the marine industry, the accumulation of living organ- as the protective coating surface deteriorates because of isms on artificial surfaces by adhesion, growth and repro- metabolic and other biological process. This makes the hull duction is known as biofouling. Biofouling is a particular surface more susceptible to corrosion and discoloration [4]. problem for underwater structures, such as pipelines, cables, In the marine industry, biofouling is a huge problem, and fishing nets, and bridge pillars. The adverse effects of ship research is focused on development of an effective anti- hull biofouling (Figure 1) include [1]: (i) Higher fuel con- fouling solution. However, the mechanism of biological sumption because the frictional resistance increased due to adhesion needs to be determined to allow development of biofouling, making the hull rougher and the ship heavier. antifouling strategies. In this review, an introduction to cur- Fuel consumption increases of about 40% have been ob- rent marine fouling strategies is given, and different kinds served because of biofouling [2], and the cost of a one-way of antifouling technologies are proposed according to the voyage from San Francisco to Yokohama has increased by adhesion mechanism. about 77% due to ship’s hull fouling [3]. (ii) More expensive and time consuming hull maintenance, because dry- docking operations need to be more frequent and longer 1 Biofouling organisms and their adhesion with marine biofouling [3]. Moreover, these cleaning pro- mechanism cesses generate a large number of toxic substances that are discharged into the ocean. (iii) Increased ship hull corrosion More than 4000 kinds of marine biofouling species have been reported globally, most of which live primarily in the *Corresponding author (email: [email protected]) shallower water along the coast and in harbors that provide

Figure 1 Heavy biofouling on the hulls of vessels. abundant nutrients [1]. In general, marine adhesion organ- larvae or spores of macrofoulers will attach to the surface. isms can be divided into two major categories. The first of Two or three weeks later, these will finally evolve into a these includes the microfouling or biofilm organisms, which complex biological community. In marine immersion ex- are bacteria and diatoms. Biofilms are ubiquitous, as long as periments, adhesion of macroorganisms usually occurs after the surfaces are exposed to water. The other category in- biofilm formation [10,13]. However, this is not always the cludes macrofouling organisms such as algae and barnacles. case [1,14], for instance the larvae of some species of bryo- The most important macro-fouling species are barnacles, zoans [15], polychaetes [16] and some other biofoulers [17] mussels, polychaete worms, bryozoans and seaweed [5]. adhere before biofilm formation. The biofouling process can be simplified as illustrated in Therefore, the process of biofouling occurs by both Figure 2. First, through a simple physical reaction, a layer physical reactions and biochemical reactions (Figure 3). The of conditioning film composed of organic materials such as physical reactions are governed by factors such as electro- protein, polysaccharide, and proteoglycan, is formed on the static interaction and water flow, and lead to formation of substrate surface. This step is short (1 min), and provides a the conditioning biofilm and adsorption of microorganisms. stickier surface for microorganisms to adhere to [6]. The biochemical reactions include EPS secretion, move- The biofilm then develops as bacteria and microalgae ment and secondary adhesion of microorganisms, formation adhere to the surface. Microorganism colonization involves of the biofilm, and adhesion of macrofoulers. Whereas the two distinct steps: reversible adsorption, and irreversible physical reactions are usually reversible, the biochemical adhesion. The former is governed mainly by physical ef- reactions are effectively irreversible. Thus, it would be eas- fects such as Brownian motion, electrostatic interaction, ier to prevent biofouling during the physical reactions rather gravity, water flow and van der Waals forces [7–10]. The than the biochemical reactions. Successful inhibition of the latter occurs mainly through biochemical effects such as physical reactions would constrain the later biochemical secretion of extracellular polymeric substances (EPS). Dur- reactions. ing biofilm formation in the marine environment, diatoms Current research on antifouling is focused on inhibition are the most important contributors. It has been reported of adhesion of diatoms and bacteria to prevent biofilm for- that microfouling alone can increase fuel consumption by mation, though such research has also encountered numer- up to 18%, and reduce the sailing speed by at least 20% ous obstacles [1]. Some of the macrofouling organisms have [11]. been also researched in detail. The adhesion strategies of the After the formation and development of the biofilm, three kinds of biofouling organisms (bacteria, microalgae,

Figure 2 Temporal settlement of fouling organisms on a substrate surface [12]. 600 Cao S, et al. Chinese Sci Bull March (2011) Vol.56 No.7

Figure 3 Biofouling process and the formation of biofilm. and macroorganisms) are discussed in sections 1.1–1.3. number of similar or homologous and mixed species, which has beneficial effects for the microorganisms [19,20]. After 1.1 Bacterial adhesion maturation of the biofilms, they disperse cells into the water to expand the species (Figure 4(b)) [21]. Bacterial adhesion occurs as a result of the interaction of The premise of the phenotypical change between re- planktonic cells with the surface by physical reactions, such versible and irreversible states is a cell density-dependent as electrostatic interactions [7], gravity [8] and water flow. system called quorum sensing. As the name indicates, the After the initial reversible absorption, bacteria use extracel- bacteria cell is able to sense that it is part of a concentration lular polymers to temporarily adhere to the surface. These of cells of a certain size (the quorum), by recognition of polymers are mainly glucose- and fructose-based polysac- specific low-molecular-weight signal compounds secreted charide fibrils [12]. The biofilm is formed when the bacteri- and accumulated by the cells in the quorum. The quorum al communities secrete more EPS (Figure 4(a)). The biofilm sensing system is important for many aspects of cell surviv- is a highly organized community usually formed by a al [22].

Figure 4 Biofilm formation. (a) SEM photographs of EPS produced by Salmonella (scale bar = 2 μm) [18]; (b) biofilm formation in five steps: initial adsorption, irreversible adhesion, maturation I, maturation II, and dispersion [21]. Cao S, et al. Chinese Sci Bull March (2011) Vol.56 No.7 601

Generally speaking, the mass of cells in biofilms ac- this has been observed in other adhesive situations [36]. counts for only 2%–5% of the total weight with the re- After the diatoms land on the surface, they actively form mainder contributed by the EPS matrix, which includes a the initial reversible attachment called primary adhesion variety of extracellular carbohydrates, proteins, nucleic acid, through secretion of EPS. The diatoms then reorient them- glycoprotein, phospholipids and other surfactants. The ratio selves and move along the surface into better positions of these various extracellular compounds excreted by vari- based on their preferences, this process is called diatom ous species is quite different [23,24], and even the same gliding. Many scientists have focused on diatom gliding in species secret different EPS compounds under different the belief that this would help to understand the adhesion circumstances [18]. Among those compounds, the polysac- mechanism. However, Holland et al. [37] found no relation charides are highly heterogeneous, containing different sorts between adhesion and locomotion of diatoms. It is generally of monosaccharide units and inorganic materials [25]. The acknowledged that diatom gliding is the result of an ac- secreted proteins, many of which are polymer-degrading tin-myosin motility system meditated by extracellular pro- enzymes, also have heterogeneous compositions, although teoglycans. Actin was first identified near the position of the there is evidence that different proteins share some common raphes (Figure 5(a), (b)), and its involvement in cell motility substances or features. For instance, the surface protein Bap has been the focus of many studies [38,39]. Later experi- [26], amyloid fibril and β-1,6-N-acetyl glucosamine [27–29], ments proved that both anti-actin drugs and antibodies to and the sequence GGDEF/EAL [18] are found in different cell surface proteoglycans inhibit diatom gliding [39,40]. proteins. However, the underlying mechanism generating force for These factors illustrate the lack of many common fea- diatom gliding is currently poorly understood. Some me- tures among different biofilms, which makes broad-spec- chanical models for locomotion have been calculated in trum antifouling a difficult target. earlier studies [41,42], and some reasonable cell locomotion mechanisms have been proposed (Figure 5(c)) [43]. After 1.2 Microalgae adhesion diatom gliding, if the diatoms continue their life cycles in that position, they will form irreversible secondary adhesion The major eukaryotic marine fouling microorganisms are by secreting a large amount of EPS [44–46]. Individual di- diatoms, fungi, and protozoan, and the dominant organisms atoms commonly generate EPS like stalks in this period are diatoms [12,13]. [45,47,48]. Diatom adhesion is a more complicated process than that EPS of diatoms is composed of carboxylated or sulfated for bacteria. Because most of the diatoms lack flagella, they acidic polysaccharides, which are involved in the primary cannot actively approach a given surface, but passively land adhesion, and proteoglycans, which are involved in diatom on the substratum. For example, benthic diatoms approach gliding and cross-linking stabilization of the biofilm matrix surfaces through the effects of either gravity [30] or water [40]. As with bacteria, EPS produced by different types of currents [31–33]. Plankton diatoms, which have almost the diatoms are diverse [49,50], and include various protein same specific gravity as seawater, land on surfaces mainly fractions and complex anionic polysaccharides with hetero- via turbulence [34]. Moreover, electrostatic interactions geneous combinations of monosaccharide [45]. In addition, such as Coulomb attraction and electrostatic contact poten- at least two types of mucilage can be detected for the same tial are also involved [35]. During contact between diatoms species of diatom [51,52]. However, some common features and a surface, van der Waals forces may also operate, and have been detected among different diatoms, such as

Figure 5 Actins in diatoms and proposed diatom locomotion mechanism. (a) Cell of Craspedostauros australis in girdle view [39]; (b) fluorescein (FITC) phalloidin stained cell of C. australis in also girdle view, which indicates F-actin along the raphe [39]; (c) proposed cell movement mechanism [43] involv- ing: (i) secretion of mucilage strands at the central pore, and their attachment to membrane components and substratum; (ii) relative backward movement of the membrane components, which leads to forward displacement of the framework itself and of the cell as a whole; (iii) breaking of the mucilage strands, which forms a short-lived trail. 602 Cao S, et al. Chinese Sci Bull March (2011) Vol.56 No.7 general structural components by time of flight-secondary are extremely important in biofouling because of their ion mass spectrometry (TOM-SIMS) [53], and modular abundance in seawater and adaptability to different envi- proteins and their supramolecular assemblies of adhesive ronments [12]. The motile spores have four flagella and no nanofibers (ANFs) [54]. These common traits could be tar- polysaccharide-rich cell wall. Typically, Ulva spores adhere geted in a method to combat diatom adhesion. to the surfaces by secreting glycoprotein, and then retract As mentioned above, we have some primary understand- the flagella and form a cell wall [62–64]. However, in some ing of the mechanisms of diatom gliding and adhesion, special conditions spores will exhibit abnormal pseudo- though the hypotheses still lack theoretical support. For settlement [65]. Freshly released glycoprotein from Ulva example, the mechanism by which the secreted mucilage spores has strong adhesion strength, and the spores cannot and actin generate force is unknown. Aspects of diatom be removed under the speed of most vessels [66]. adhesion are even more uncertain, such as how it is initiated Protein adhesives from algae, mussels, and polychaetes or inhibited, and cannot be explained by the established have several features in common, including high contents of theory of mucilage secretion. lysine, glycine and serine, and extensive polypeptide repeats with abundant dihydroxyphenylalanine (DOPA) side-chains, 1.3 Macro-organism adhesion which will displace water molecules to facilitate strong adhesion [25,67]. However, they are quite different from the The most problematic biofouling effects arise because of barnacle adhesives [68,69]. colonization of macroorganisms such as spores of macro algae, barnacle larvae, bryozoans, molluscs, polychaete, tunicates, and coelenterates [12]. 2 Antifouling methods A common trait of macroorganism settlement is biofilm cueing. As early as 1963, it was shown that the increased The severity of biofouling depends on a large number of diatom concentrations could induce maturation of barnacle parameters, including temperature, salinity, light, geography, larvae [55]. However, subsequent experiments have shown depth, and voyage speed [1,70]. For example, biofouling is that biofilms do not facilitate settlement of all macroorgan- generally more serious in areas with high water temperature isms. This has been demonstrated in a variety of marine because this is the principal condition determining breeding organisms such as barnacles, oysters, and Ulva [25] fed by periods and rates of growth of biofouling organisms [71]. bacteria [56] and diatoms [57]. Because of competition for Unfortunately, these important factors cannot be modified nutrients and light, it is believed that microorganisms pro- to a large extent. Therefore, to effectively prevent biofoul- duce metabolites to repel specific macroorganisms [58]. ing, a variety of coatings have been investigated to chemi- Another common characteristic of macroorganism set- cally inhibit the fouling organisms. From another point of tlement is physiochemical cueing. Some experiments have view, during the development of microtechnology, the in- shown that invertebrate larvae seem to be able to select fluence of microscale physical factors on biofouling has suitable substrates, and their choice is determined by sur- been researched and some new antifouling technologies face topography, water streaming conditions and chemical have arisen that involve changing physical factors. In addi- properties [25]. Moreover, it is generally accepted that lar- tion, microorganisms can secrete metabolites that inhibit the val settlement and metamorphosis are influenced by chemi- attachment of some macroorganisms [72], and concentrated cal cues originating from conspecific adults, prey organisms, extracts of these metabolites could also be effective anti- and substrates [59]. fouling agents. Though the settlement of macroorganisms follows the In summary, antifouling methods can generally be di- above common guiding cues, adhesion mechanisms are vided into three categories: chemical, physical, and biolog- quite different in specific organisms. Barnacles and Ulva ical methods. have been investigated as representatives of invertebrate and macroalgae groups, respectively. The settlement mech- 2.1 Traditional chemical methods anism of barnacles has been studied in great detail, and results indicate that the cyprid antennule consists of four Biofouling has been recognized as problematic for more segments that are responsible for crawling, attachment and than 2000 years [73], and many kinds of antifouling meth- sensory functions [60]. When an appropriate surface is ods have been investigated over this time [1]. Since the late found, the cyprid will adhere by secretion of granulated 20th century, organic tin and its derivatives have been cement containing high concentrations of proteins. This widely used as antifouling coatings because of their activity cement embeds the antennular attachment organs and hard- against a wide range of fouling species. Organotin com- ens because of protein polymerization. After stable settle- pounds that have been used as antifoulants include tribu- ment, cyprids metamorphose into juvenile barnacles, and tyltin oxide (TBTO), and tributyltin fluoride. Those anti- finally become adults [61]. fouling organotin compounds are powerful fungicides, Ulva spores are another well-studied macroorganism, and and will completely inhibit the growth of most fouling Cao S, et al. Chinese Sci Bull March (2011) Vol.56 No.7 603 organisms at a very low concentration [74]. The paints con- oxidation, its mechanical properties are inferior to those of taining these compounds can be classified as those with insoluble matrix coatings. insoluble and soluble matrices, according to the chemical characteristics of the binder and their water solubility. 2.2 Modern chemical antifouling methods Insoluble matrix antifouling paints have a polymer matrix (such as vinyl and epoxy) that will not erode in water (i) Tributyltin self-polishing copolymer coatings. As noted [1]. When the coating is immersed in seawater, the soluble in section 2.1, both insoluble and soluble matrix antifouling toxic materials dissolve, which leaves a multiporous struc- coatings have their deficiencies. Consequently, alternative ture known as the leached layer. Seawater then penetrates coatings have been investigated. In 1974, Milne and Hails deeper into the film and more poisonous materials dissolves patented the first TBT self-polishing copolymer (TBT-SPC) in the water. The advantage of this kind of paint is that the technology, which provided an excellent antifouling effect structures are mechanically strong and stable to oxidation that revolutionized the entire shipping industry [1]. and photodegradation. Thus the coatings can be made very TBT-SPC paints are based on acrylic polymer (usually thick to increase the content of toxic materials. However, at methyl methacrylate) with TBT groups bound to the poly- some stage the leached layer will be so thick that water mer backbone by an ester. When immersed in water, the cannot penetrate any deeper, and the rate of release will fall soluble pigment particles (such as ZnO) begin to dissolve under the minimum value required for antifouling (Figure [77]. The polymer of TBT methacrylate and methyl meth- 6(a)). Therefore, the lifespan of insoluble matrix antifouling acrylate is hydrophobic, which prevents water from infil- paints is as short as 12–18 months [75]. trating the paint film. Therefore, water can only fill the To lengthen the lifespan of antifouling coatings, soluble pores generated by the dissolution of soluble pigment parti- matrix antifouling coatings were developed. As implied by cles. Moreover, the carboxyl-TBT linkage is easily hydro- the name, both the toxic materials and matrix, which con- lyzed in slightly alkaline environments such as in seawater tains a great amount of resin, can dissolve in seawater. In (pH 7.5–8.5). This results in cleavage of the TBT portion this case, the leached layer can be much thinner and toxic from the copolymer, and releases the biocides into the water materials deeper in the film can be easily exposed to water, [74]. Once many TBT portions have been cleaved, the par- which lengthens the lifespan of the antifouling coating tially reacted brittle polymer backbone can be easily washed (Figure 6(b)) [1]. The release rate will exponentially in- off by the moving seawater, which exposes a fresh coating crease as the sailing speed increases. However, during the surface (Figure 7(a),(b)) [74,78]. The hydrolysis process static conditions that favor settlement of fouling organisms provides a low hull roughness (about 100 μm), so as not to the pores of this coating can become blocked by insoluble increase the resistance of the ship’s hull [1]. salts, which greatly reduces the release of biocides [76]. In One of the major advantages of an antifouling coating addition, because of the resin’s brittleness and instability to such as this is that manipulating the polymer chemistry can

Figure 6 Method of release and biocide release rates of (a) insoluble matrix paints and (b) soluble matrix paints [1].

Figure 7 TBT-SPC system and cross-sectional SEM image. (a) Self-polishing TBT copolymer system [74]; (b) SEM image showing cross-section of TBT-SPC (magnification 5000) [78]. 604 Cao S, et al. Chinese Sci Bull March (2011) Vol.56 No.7 control the polishing rate. Therefore, it is possible to bal- Polymer—COO—Na+(solid) + X－+ Zn2+. ance high effectiveness and a long lifespan, and the coatings The Zn2+ is discharged into water for antifouling, and the can be customized for ships operating under different con- soluble acidic polymers can be washed from the surface. ditions, such as duration of their idle periods and sailing Beside zinc, the majority of tin-free antifouling paints cur- speed [1]. It has been shown that release rate of TBT in rently available contain copper, and some contain silver seawater is almost constant with sailing speed, and thus [81]. Currently, the major copper compounds used for anti- high antifouling performance can be obtained even if the fouling include metallic copper, cuprous thiocyanate, and ship is not moving. In addition, the maintenance is conven- cuprous oxide [82,83]. Copper ions as Cu2+ have a major ient and low cost. Moreover, TBT-SPC paints have high role in antifouling [1]. mechanical strength, high stability to oxidation, and short Compared with the broad-spectrum TBT antifouling drying times [74]. The unavoidable formation of biofilm coatings, copper-containing coatings can only target specif- does not largely affect the net biocide leaching and binder ic fouling organisms. Biological indicators differ widely reaction rates [79]. Therefore, in general, the TBT-SPC an- with respect to copper sensitivity, and a general decreasing tifouling coating was widely applied in the shipping indus- order of sensitivity would be: microorganisms > inverte- try before it was banned. brates > fish > bivalves > macroalgae [84]. Therefore, some (ii) Tin-free SPC technology. As discussed edrlier, booster biocides that are highly toxic to macroalgae, barna- TBT-SPC coatings have many advantages as antifouling cles, and bryozoans are added to improve the antifouling coatings. However, the damaging effect of TBT on non- proprieties. These biocides include Irgarol 1051 and Diuron target organisms cannot be ignored. Consequently, TBT has [83,85], copper pyrithione and isothiazolone [1,86]. been restricted as of the International Maritime Organiza- Undoubtedly, there is concern about the influence of tion (IMO) conference in 1998, and these coatings have copper containing coatings and booster biocides on the ma- been banned from 1 January, 2008 [80]. Therefore, TBT- rine environment [85]. However, a number of scholars have free systems have been commercially introduced. highlighted that the boosters are biodegradable materials Generally speaking, the tin-free antifouling coatings can with short half-lives. Unfortunately, the heavy metals do be divided into two categories: controlled depletion systems bioaccumulate in the internal organs of marine life [87]. (CDPs), and tin-free self-polishing copolymers (tin-free Generally speaking, there is no simple and nontoxic solu- SPCs). The former coatings upgrade the traditional soluble tion for the biofouling problem [88]. However, copper con- matrix technology by incorporating modern reinforcing taining coatings are considered as a transition between toxic resins with the same antifouling mechanism as the conven- and non-toxic coatings. tional resin matrix paints. The latter coatings function in a (iii) Non-toxic antifouling technology. Although in the similar manner to TBT-SPC but do not contain tin. Cur- short term, no alternative antifouling technology seems ca- rently, these two types of paints are produced by many pable of replacing biocide-based coatings, there are some companies, including Ameron, Chugoku MP, and Hempel’s non-toxic technologies that have been developed. MP [1]. The differences between CDPs and tin-free SPCs For instance, non-stick fouling-release compounds (such are illustrated in Table 1, and from these parameters we can as silicone coatings) have been trialed for antifouling see the performance of tin-free SPCs is better than the through release of macrofouling organisms when hydrody- CDPs. namic conditions are sufficiently robust [1,37]. In this case, Tin-free SPCs react in a similar to organic tin SPCs, but it appears that fluoropolymers and silicones possess the their matrix material is mostly acrylic copolymer and necessary properties for antifouling by release [1]. Some non-tin metals such as copper, zinc, and silicon. For exam- low surface energy coatings have also been prepared with ple, the Exion series from Kansai Paint [1] uses insoluble modified acrylic resin and nano-SiO [89]. However, accu- Zn acrylate, which hydrolyzes to soluble acidic polymer. 2 mulated fouling organisms are not as easily released as an- The following reaction is assumed: ticipated [37,90,91]. In addition, this method has many de- + Polymer—COO—Zn(solid)—X + Na → ficiencies, such as high cost, poor mechanical properties,

Table 1 Contrast of performance of CDPs and tin-free SPCs

CDPs Tin-free SPCs Self-polishing Poor Smooth paint surface during sailing Leached layer Thick Thin and stable Biocide release Hard to control, not constant Continuous and constant at the same velocity and sea water conditions Idle periods Little activity High activity Lifetime duration Short, up to 3 years Long, 5 years Maintenance High cost, sealer coating needed Low cost, re-coating directly

Cao S, et al. Chinese Sci Bull March (2011) Vol.56 No.7 605 and the difficulty of recoating. Therefore the antifouling In the case of microfouling, the process is more compli- performance is limited, which leads to increased interest in cated [98,103], because polysaccharide-based adhesives are other methods. Generally speaking, non-toxic antifouling as important as proteins during secondary adhesion. Gener- technology research can explore either biological or physi- ally, polysaccharide degradation is executed by glycosylase. cal methods. However, to degrade polysaccharides is difficult because Biological methods involve using a variety of enzymes the process is quite complex [106] and glycosylase can tar- or metabolites secreted by cells as substitutes for traditional get only a limited range of linkages. Consequently, it would biocides [23]. Because these organic secretions are biode- be difficult to choose an appropriate glycosylase for broad- gradable, they should be environmentally friendly [25]. spectrum antifouling [103]. Physical methods include electrolysis, radiation, and other physical methods to reduce biofouling. They can also 3.2 Enzymes that disrupt the biofilm matrix utilize modification of surface physical properties, such as topography or charge potential, to minimize adhesion Because of the variety of EPS, biofilms are very complex strength [1]. and the disintegration of their polymeric networks would require very broad combinations of both hydrolases and lyases [25]. In addition, because biofilms are very adaptable 3 Biological methods to external conditions, the degradation of the crucial com- ponent will induce the generation of alternative components As mentioned in earlier, some organisms can secrete en- that will replace the original and establish a new network to zymes or metabolites to inhibit the growth of their competi- proliferate the organisms [107]. This means that the biofilm tors. These secretions have low-toxicity and are biode- will not disintegrate. Tests have shown that though alginase gradable, and have received much attention in recent years. could detach a thin biofilm [108], it had no effect on an Researchers have attempted to extract high concentrations identical biofilm that was already fully established [107]. of these secretions to use for biological antifouling. As early In summary, because of the complexity and adaptability as 1999, it was reported that the active substances secreted of the biofilm, the antifouling method of disrupting the bio- by blue algae could inhibit the growth of diatoms [92]. film matrix may not be suitable and effective. Functional antifouling components have also been discov- ered in other organisms such as fungi [93], sponge [94] and 3.3 Enzymes that generate deterrents and biocides some other bacteria [95,96]. The application of enzymes as antifouling agents has In recent years, antifouling research has focused on the ex- been successfully investigated recently. Many types of en- traction of metabolites secreted by different marine animals zymes, such as oxidoreductases, transferases, hydrolase, or plants that have strong antifouling ability. Such antifoul- lyase, isomerase, and ligase, have been reported to have ing compounds should be regarded as deterrents rather than antifouling capabilities [25,58,97–106]. From the perspec- toxins [23,25]. tive of enzymatic antifouling technology, biofouling prob- Some of the enzymes that have such effect include glu- lems are caused by the formation and reproduction of bio- cose oxidase, hexose oxidase, and haloperoxidase [25,99]. films, and the adhesion of spores and larvae of macroorgan- For instance, glucose and hexose oxidase is used to generate isms. Therefore, the functions of enzymes for antifouling hydrogen peroxide to induce oxidative damage in living applications can be divided into the following four catego- cells [109], and haloperoxidase catalyses the formation of ries: degradation of adhesives used for settlement, disrup- hypohalogenic acids usually used in water treatment sys- tion of the biofilm matrix, generation of deterrents/biocides, tems as disinfecting agents [25]. In addition, hydrogen per- and interference with intercellular communication. oxide will decompose into water and oxygen, and the rate of this process is quite high in seawater [110]. Hypohalogenic 3.1 Enzymes that degrade adhesives used for settlement acids have similar characteristics, and could also be investigated in future as nontoxic and biodegradable antifouling In the case of macrofouling, proteins and proteoglycans substances [99]. have a dominant role in the adhesion process. As is widely known, proteases can hydrolyze peptide bonds at different 3.4 Enzymes that interfere with intercellular commu- sites, and this kind of enzyme can be used to degrade muci- nication lage based on peptide and hence prevent biofouling. For example, the attachment of Ulva spores, barnacle cyprids As discussed in section 1.1, quorum sensing has an im- and bryozoans can be effectively inhibited by serine prote- portant role in the formation of biofilms. Studies have ase [58,98]. It has been confirmed that this inhibition is shown that N-acyl homoserine lactones (AHL) are required caused by reduction of adhesive effectiveness rather than for quorum sensing by some Gram-negative bacteria [22]. any toxic or deterrent effect [105]. Therefore, elimination of AHL may thus prevent the 606 Cao S, et al. Chinese Sci Bull March (2011) Vol.56 No.7 development of bacterial fouling [25]. AHL acylase colors, which affect the attachment and growth of spores degrades AHL, and as the concentration of this enzyme and worms [118,119]. increases, biofilm formation is inhibited and the settlement of Ulva spores and polychaete larvae is affected [110,111]. 4.2 Antifouling by modification of surface topography Consequently, AHL acylases can also inhibit the settlement and hydrophobic properties of macroorganisms to some extent. As stated in section 1.1, biofilm is mainly formed by microorganisms, including bacteria and microalgae, which are 3.5 Challenges for enzymatic antifouling methods approximately 1–100 μm in size. At this scale it is possible The seawater temperature ranges from –2°C to 30°C, which to precisely modify the surface microstructure. Therefore, in can largely affect enzyme catalytic activity and stability. In recent years, varying surface characteristics, including sur- addition, each enzyme will itself decompose if the tempera- face roughness, topography, hydrophobic behavior, and ture is too high, and then the lifespan of the enzymatic anti- lubricity, have been investigated for antifouling application fouling coating will also decrease. Therefore, balancing of [120]. To simplify the models used in these studies most the effectiveness and lifespan will be a major challenge. researchers regard diatoms or bacteria as representative ad- Furthermore, the design of an appropriate coating matrix to hesion organisms. contain the enzymes will be another crucial step for suc- Because of material properties, surface wettability has a cessful application [112]. In addition, the distribution of large impact on the adhesion of biofouling organisms. Study enzyme and its amount should also be analyzed in detail, be- has shown that fouling diatoms [37] adhere more strongly to cause soluble enzymes will soon form a thick leaching layer. a hydrophobic polydimethylsiloxane (PDMSE) surface than to glass. For other fouling organisms such as bacteria and Ulva spores, if the surface contact angle is greater than the 4 Physical antifouling methods adhesion is stronger [121–124]. However, the strength of attachment of Ulva spores is greater to hydrophilic than 4.1 Antifouling by electrolysis and radiation hydrophobic surfaces [66,125]. Moreover, hydrophilic sur- Many methods for physically preventing biofouling have faces are thought to be capable of antifouling. For example, been investigated. Among them, the most common method antifouling behaviors are exhibited when adding metal na- is to produce hypochlorous acid (HClO), ozone bubbles, noparticles such as TiO2, because the photocatalytic activi- hydrogen peroxide or bromine through electrolysis of sea- ties introduced by solar ultraviolet will make the surface water [1,113,114]. Because of their strong oxidizing ability, more hydrophilic so that the formed biofilm is washed more HClO and other compounds will spread all over the ship’s easily [126]. However, some species studied have exhibited hull and eliminate areas of fouling organisms. However, opposite adhesion behavior on the same sets of surfaces, some of these systems are not highly efficient because of a highlighting the importance of differences in cell-surface large voltage drop across the surface, and they will intensify interactions [32,66]. Therefore, the differences in settlement the corrosion problems of steel. Consequently, titanium- and adhesion behavior have inspired the development of a supported anodic coating has been suggested because of its surface that presents both hydrophilic and hydrophobic do- advantages such as having low decomposition tension, mains to settling (attaching) cells and organisms. Different higher current efficiency, lower energy consumption [115], patterns of such surfaces have also been evaluated [127, although the development of this has been limited. Anti- 128]. fouling could also be achieved by microcosmic electro- Surface topography also affects the adhesion of fouling chemical methods, which are based on direct electron organisms. It has been shown that rougher surfaces increase transfer between an electrode and the microbial cells. This adhesion of Pseudomonas [129]. Furthermore, Scardino et causes electrochemical oxidation of the intercellular sub- al. proposed the attachment point theory (Figure 8), which stances, but is expensive and the efficiency has not been indicates that more attachment points during adhesion re- established [25]. sults in stronger more prolific adhesion [130,131]. This the- The antifouling abilities of vibration methods, such as ory is consistent with the settlement of barnacle cyprids acoustic technology, have also been confirmed [116]. Hy- [132]. droids, barnacles and mussels can be inhibited to some ex- The effects of feature size, geometry and roughness on tent by either external vibration sources or piezoelectric the settlement of zoospores of Ulva were evaluated using coatings [117]. However, the huge power consumption of engineered micro topographies in PDMSE. The results these methods is difficult to overcome. identified an engineered roughness index (ERI) that can Finally, other studies have evaluated magnetic fields, ul- influence antifouling [133]. ERI could be calculated as: traviolet radiation and radioactive coatings [1], but these ERI=(r*df)/fD, based on Wenzel’s roughness factor (r), the methods are not practical in application. An additional po- depressed surface fraction (fD), and the degree of freedom of tential method involves the use of substrates with different spore movement (df). Different surface topographies (Figure Cao S, et al. Chinese Sci Bull March (2011) Vol.56 No.7 607

Figure 8 Attachment point theory experiments, which illustrate that the adhesion strength is highest in (a) and (b) (multiple attachment points) and lowest in (d) (least attachment points) [130].

9(a)) have different ERIs, and that with the largest ERI cated and influenced by many physical reactions, while (Sharklet AF™) will perform most effectively as an anti- electrostatic interactions are expected to have a major role foulant. It was proposed that the responses were governed [138]. Therefore, potentials of the surfaces or microfouling by the same underlying thermodynamic principles as wetta- organisms have been measured to determine the relationship bility [134]. The Sharklet AF surface was also tested with between them. other organisms such as Staphylococcus aureus (Figures 9(b) It is well known that bacterial cell surfaces possess net and (c)) and appeared to have effective antifouling perfor- negative electrostatic charge because of ionized phosphoryl mance [135]. The effectiveness of designed nanoforce gra- and carboxylate substituents on outer cell envelope macro- dients for antifouling applications was tested with Ulva, and molecules that are exposed to the extracellular environment. the results showed the surfaces with nanoforce gradients The influence of peripheral electronegativity can be as- ranging from 125–374 nN all greatly reduced spore settle- sessed based on the measurement of zeta potential (Figure ment [136]. 10(a)), which is most often determined by estimating the Multivariate methods were used to identify relationships electrophoretic mobility of cells in an electric field [139]. between bacterial attachment, water transport, and the sur- ZPs of many microorganisms have been measured in dif- face properties of modified polysulfone (MPS) membranes, ferent physiological states [140], such as for Chlorella which showed that the interrelations were quite complicated (ZP=–17.4 to –19.8 mV, independent of pH from 4–10), [137]. In summary, the research into modification of surface diatom Nitzschia (ZP=–28 mV, stationary phase) [141], and topography to achieve antifouling is still at the experimental Pseudomonas sp. (ZP=–46.9 mV) [140]. Microorganisms stage. The identification of effective antifouling topogra- are negatively charged over a large pH range (Figure 10(a)), phies typically occurs through trial-and-error rather than and in natural sea water (pH 7–8) will carry a negative predictive models. Although some empirical principles such charge [142]. As a result, electrostatic interaction between as attachment point theory and the ERI index have been cells and metal cations promotes the adhesion process. proposed, these theories are not sufficient to explain the real In some other industries, varying the pH of the suspen- situation. Therefore, these formulas are not expected to sion and hence shifting the surface charges of the bacterial guide the development of antifouling methods. cells has been used as an effective method to reduce biofouling [143]. However, it is impractical in the case of marine fouling. Consequently, attempts have been made to 4.3 Antifouling by changing the zeta potential modify the charge potential of the surfaces to inhibit the As mentioned in section 1, microbial adhesion is compli- cells from attaching to them to some extent. Fouling by

Figure 9 Spore attachment on surfaces with different topographies. (a) SEM images of engineered topographies on PDMSE surface and corresponding spore settlement data [133]. Uniform surface of circular pillars (ERI=5.0) or wide ridges (ERI=6.1) reduced settlement by more than 30%. A multi-featured topography consisting of pillars and equilateral triangles (ERI=8.7) reduced spore settlement by 58%. The largest reduction 77%, was obtained with the Sharklet AF topography (ERI=9.5). SEM images of S. aureus on smooth PDMSE (b) and Sharklet AF surface (c) after 21 d [136]. 608 Cao S, et al. Chinese Sci Bull March (2011) Vol.56 No.7

Figure 10 Zeta potential with diatom cells. (a) Cross-section representation depicting the various solvent layers surrounding a bacterial cell [139], and zeta potentials of two picocyanobacterial strains as a function of pH [142]; (b) fouling coverage of P. aeruginosa vs. zeta potential of nylon membranes after various periods of time [129].

Pseudomonas aeruginosa was minimal when the surface without biofilms. Thus, it is important to research ways to charge was minimized, and increased with increasing prevent biofilm formation. Because diatoms are the domi- charge (positive or negative) (Figure 10(b)) [129]. Ulva nant organisms in biofilms, they have been widely evaluat- spores were demonstrated to have a reduced tendency to ed in a variety of experiments as a representative of micro- settle on negatively charged surfaces of polytetrafluoroeth- organisms. ylene (PTFE) [64]. Many other phenomena observed during Many kinds of antifouling technologies have been inves- microorganism settlement on self-assembled monolayers tigated to counteract the marine foulers. For instance, [121,144] can be partly explained by electrostatic interac- TBT-SPC is one of the most effective antifouling methods. tions [145,146]. Discharge treatment can be used to create However, biocide-based antifouling coatings are environ- carboxylic acid groups on polymer surfaces [147], which can mentally unsuitable, and research on non-toxic coatings is also be discussed in terms of the repulsive electrostatic interac- of importance. This research generally focuses on physical tions [148]. methods, including electrolysis and radiation, modification of surface structures and wettability, or change in the charge 4.4 Challenges for physical antifouling methods potential of surfaces. The alternative is biological methods, including using enzymes or metabolites secreted by cells to In summary, in most cases there are no convincible theories replace toxic biocides. to explain different antifouling performance by modification In the case of biological methods, the current situation of surface zeta potential, topography, and wettability. Con- shows that enzymes degrading adhesives (proteases and sequently, many experiments investigating various parame- glycosylases) and enzymes interfering with intercellular ters are insufficient for solving biofouling problems. De- communication (acylase) can effectively prevent biofouling. tailed information on the biological adhesion mechanism is The former approach is mainly targeted at macroorganisms required as the biofouling process is a complex biochemical and the latter at microorganisms, and some kind of combi- problem rather than a pure physical problem. nation of both enzymes will possibly produce a better result. Further documentation of trials on marine crafts should be obtained, and the various enzymes should be direct tested 5 Conclusions after design and manufacture of enzymatic coatings. In the case of physical methods, broad-spectrum anti- In general, biofouling on marine structures is characterized fouling could be performed. Currently, the most successful by two critical events: the formation of biofilms, which in- method is the modification of surface topography, and cludes reversible physical adsorption, irreversible secondary Sharklet AF has demonstrated to be effective against a vari- adhesion, and then proliferation of the microorganisms; and ety of fouling organisms. However, the understanding of the settlement and growth of spores or larvae of macroor- such mechanisms is still very poor. Although some empiri- ganisms. In most cases, it is easier for macroorganisms to cal theories such as attachment point theory and the ERI settle on specific biofilms through cueing than on the surface principle have been proposed, there is a lack of convincing Cao S, et al. 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